Trophic transfer of polyunsaturated fatty acids across the aquatic–terrestrial interface: An experimental tritrophic food chain approach

Abstract Aquatic and their adjacent terrestrial ecosystems are linked via the flux of organic and inorganic matter. Emergent aquatic insects are recognized as high‐quality food for terrestrial predators, because they provide more physiologically relevant long‐chain polyunsaturated fatty acids (PUFA) than terrestrial insects. The effects of dietary PUFA on terrestrial predators have been explored mainly in feeding trials conducted under controlled laboratory conditions, hampering the assessment of the ecological relevance of dietary PUFA deficiencies under field conditions. We assessed the PUFA transfer across the aquatic–terrestrial interface and the consequences for terrestrial riparian predators in two outdoor microcosm experiments. We established simplified tritrophic food chains, consisting of one of four basic food sources, an intermediary collector gatherer (Chironomus riparius, Chironomidae), and a riparian web‐building spider (Tetragnatha sp.). The four basic food sources (algae, conditioned leaves, oatmeal, and fish food) differed in PUFA profiles and were used to track the trophic transfer of single PUFA along the food chain and to assess their potential effects on spiders, that is, on fresh weight, body condition (size‐controlled measurement of nutritional status), and immune response. The PUFA profiles of the basic food sources, C. riparius and spiders differed between treatments, except for spiders in the second experiment. The PUFA α‐linolenic acid (ALA, 18:3n‐3) and ɣ‐linolenic acid (GLA, 18:3n‐6) were major contributors to the differences between treatments. PUFA profiles of the basic food sources influenced the fresh weight and body condition of spiders in the first experiment, but not in the second experiment, and did not affect the immune response, growth rate, and dry weight in both experiments. Furthermore, our results indicate that the examined responses are dependent on temperature. Future studies including anthropogenic stressors would deepen our understanding of the transfer and role of PUFA in ecosystems.


| INTRODUC TI ON
Natural ecosystems are in exchange with neighboring ecosystems. Aquatic and adjacent terrestrial ecosystems are linked via the exchange of organic and inorganic matter (Baxter et al., 2005;Schindler & Smits, 2017). Leaves and terrestrial invertebrates falling into streams can be an important subsidy for stream food webs (Baxter et al., 2005;Wallace et al., 1997). In turn, emergent aquatic insects can be a food source for terrestrial predators, such as bats, lizards, and spiders (Kato et al., 2004;Sabo & Power, 2002;Sullivan et al., 1993). In particular, when terrestrial food sources are scarce, for example, in spring, terrestrial predators can benefit from feeding on emergent aquatic insects (Nakano & Murakami, 2001;Wesner, 2010). Besides food quantity, food quality is key for understanding energy fluxes between ecosystems and for predicting effects on subsidized food webs (Marcarelli et al., 2011).
Emergent aquatic insects are considered high-quality food for terrestrial predators, because they contain up to 10 times more longchain (≥20 carbon atoms) polyunsaturated fatty acids (PUFA) than terrestrial insects (Hixson et al., 2015;Parmar et al., 2022). Aquatic primary producers, such as diatoms or cryptophytes, can synthesize long-chain PUFA that are subsequently available to aquatic consumers and transferred across trophic levels (Ahlgren et al., 1992;Kainz et al., 2004;Strandberg et al., 2015). In contrast, terrestrial primary producers typically do not produce long-chain PUFA (Sayanova & Napier, 2004). Most consumers are incapable of synthesizing longchain PUFA de novo and thus rely on an adequate dietary supply with these essential compounds (Twining, Bernhardt, et al., 2021).
The transfer of PUFA from aquatic into adjacent terrestrial ecosystems via emergent aquatic insects may thus strongly influence riparian food webs. Due to their important role in physiological processes, long-chain PUFA are typically stored in tissues without greater modifications. Thus, PUFA tend to bioaccumulate and are nearly twice as efficiently transferred to the next trophic level as bulk carbon (Arts et al., 2001;Brett & Müller-Navarra, 1997;Gladyshev et al., 2013).
Previous studies examined the PUFA profiles of aquatic insects (Martin-Creuzburg et al., 2017;Moyo et al., 2017;Scharnweber et al., 2020), the PUFA transfer via aquatic insects to terrestrial predators Twining et al., 2019;Twining, Parmar, et al., 2021), and effects of dietary PUFA on terrestrial predators in the field (Fritz et al., 2017) as well as under controlled laboratory conditions (Mayntz & Toft, 2001;Twining et al., 2016Twining et al., , 2019. For the latter, it remains open to which extent the results can be transferred to the field because environmental factors such as temperature can also affect the performance of predators, for example, regarding growth (Brown et al., 2004) and immune function (Wojda, 2017).
Our aim was to examine how PUFA are transferred along experimental tritrophic food chains and to explore potential effects on a terrestrial predator. The latter was studied in outdoor microcosms under virtually realistic environmental conditions, that is, the spiders were exposed to normal weather conditions and had the possibility to build orb webs on nettles like in their natural habitat. Overall, we conducted two outdoor microcosm experiments each with basic food sources, the Chironomus riparius (Diptera, Chironomidae) and the spider Tetragnatha sp. in a food chain. Tetragnatha sp. are suitable model organisms to study direct and indirect effects of PUFA in aquatic-terrestrial food webs because they commonly occur in riparian areas, consume aquatic emergent insects (Kato et al., 2004), and capture their prey with orb webs (Reitze & Nentwig, 1991).
Furthermore, it was shown that spiders aggregate in riparian areas during peak emergence of aquatic insects (Henschel et al., 2001;Paetzold et al., 2011) and potentially serve as prey for other organisms such as birds (Poulin et al., 2010), which may result in a PUFA transfer via spiders to higher trophic levels.
The food chains varied in the basic food source, which were selected to represent different PUFA profiles enabling us to determine potential differences in their transfer and effects on spiders.
Therefore, PUFA profiles of the basic food sources, C. riparius, and spiders were analyzed. Additionally, we recorded fresh and dry weight, growth rate, body condition (size-controlled measurement of nutritional status), and immune response of spiders to assess potential effects of PUFA on spider performance. We expected (1) different PUFA profiles between treatments at all trophic levels of the food chain, because differences in PUFA profiles of the basic food sources can propagate along the food chain; (2) temporal changes in PUFA profiles of the spiders during the experiment, due to the fact that PUFAs are assimilated after a certain time into the tissue of organisms; and (3) treatment differences in the spiders' fresh and dry weight, growth rate, body condition, and immune response due to the differences in PUFA profiles of the chironomids consumed by the spiders.

| Spider and nettle collection
For the outdoor microcosm experiments, spiders of the genus Tetragnatha were collected at pristine streams (49°16′13″N, 8°2′54″E and 49°15′43″N, 7°57′36″E) in the Palatinate Forest Nature Park, a forested low mountain range in Germany. The collection for the first experiment was conducted on the 17th, 23rd, 24th, and 25th of April 2019 and for the second experiment on the 17th, 19th, and 24th of June 2019. Until the start of the experiment, the spiders were kept in climate chambers at 20°C and were fed once a week with one adult C. riparius raised in standard cultures (OECD, 2011).
Feeding frequency varies between and within spider species (Foelix, 2011) and the immune response of male and pregnant female spiders can be highly variable (Ahtiainen et al., 2005(Ahtiainen et al., , 2004. Therefore, whenever possible, only female, adult and nonpregnant (checked visually) spiders of the species T. montana were used in the experiment to minimize variability in their feeding and immune response (Table S1).
Nettles, Urtica dioica, were used in the microcosms because they are common along streams (Davis, 1989) and are frequently used by spiders in their natural habitat to build their orb webs.

| Chironomidae and basic food sources
Adult C. riparius, hereafter chironomids, were used as food for the spiders. Chironomids were gathered from laboratory cultures that were maintained based on the OECD-guideline 235 (OECD, 2011); they were cultured in glass vessels (30 cm × 20 cm × 10 cm) with a layer of silica sand (height ~ 0.1 cm) in a climate chamber (20 ± 2°C) with a 16-h light (~1000 lux) and 8-h darkness light cycle.
Chironomid cultures differing in the food source for chironomid larvae, termed as basic food sources below, were set up at the end of February 2019 to gain organisms with specific PUFA profiles. The early setup of the cultures was made, to ensure that at the beginning of the experiment, the chironomids had only consumed the specific basic food sources during their larval stages. Overall, four basic food sources were used and later the treatments of the experiments are named after the basic food sources.
The latter three were ground before being fed to the chironomids. Fish food is used in the OECD-guideline 235 (OECD, 2011) to feed chironomids as it comprises a suitable nutrient composition for chironomids. Furthermore, fish food includes ingredients with aquatic and terrestrial origin among others fish and cereals and hence should have a broad PUFA profile. Algae was used as aquatic food source and contained phytoplankton and minerals, which usually is enriched in n-3 PUFA in comparison with n-6 PUFA (Strandberg et al., 2015). In contrast, oatmeal represented a typical terrestrial food source, because it contains high levels of LIN and only small-to-no amounts of ALA and EPA (Torres-Ruiz et al., 2010).
Conditioned leaves were considered as semiaquatic food, because they originate from terrestrial plants, but are modified by aquatic organisms. Conditioned leaves can be an important food source for aquatic invertebrates, especially in small order streams (Graça & Canhoto, 2006;Vannote et al., 1980). Conditioned leaves are colonized by microorganisms such as aquatic fungi and bacteria as well as eukaryotic microalgae. Particularly, aquatic fungi can enhance the protein and lipid content of leaves and decompose otherwise hardly degradable leaf compounds, such as cellulose, rendering leaves more nutritious and digestible (Bärlocher, 1985).

| Experimental design
We conducted two experiments on Campus Landau (49°12′15″N, 8°6′27″E) in southwest Germany. The first experiment took place from the 29th of April to the 12th of June 2019, and the second experiment was run from the 8th to 29th of July 2019. The duration of the two experiments differed, because during the second experiment, the mortality of spiders was elevated, probably due to very hot temperatures inside the microcosms of up to 32 ± 8°C ( Figure S1).
Every treatment consisted of one food chain with a basic food source at the lowest trophic level, followed by chironomids as the second level and spiders as the highest trophic level. The treatments differed only in the basic food source supplied to the chironomid larvae ( Figure 1). The first experiment included algae, fish food, and oatmeal as basic food treatment, whereas the second experiment included leaves, fish food, and oatmeal. The basic food treatments differed between experiments due to limited labor capacity preventing us to run all treatments simultaneously. Per treatment, 20 replicates were used. Every replicate consisted of one spider and one nettle in a microcosm (60 × 60 × 90 cm aerarium). The spiders and nettles were assigned randomly to the microcosms.
Spiders were fed on 2 days per week with a tweezer to ensure the spiders consumed the chironomids. In the first experiment, spiders were fed two chironomids per week and in the second experiment four chironomids per week. The number of chironomids differed because the algae-based chironomid cultures were less productive during the first experiment and therefore prohibited feeding the spiders with four chironomids, which presumably better matches the energy needs of spiders. For the first 2 weeks, dead spiders were replaced in both experiments. Before the replacement spiders were put in the microcosms, they were being fed with the total amount of chironomids the dead spider had consumed and over the same period the dead spider was fed to safeguard that the results are not influenced by different food quantities.

| Weekly measurement of fresh weight, growth and body condition
Once per week, the spiders were weighed. This fresh weight was used to calculate the growth rate (g week −1 ) of the spiders, where t x is the number of weeks of the experiment and t 0 the start of the experiment: Furthermore, a picture of every spider was taken on top of millimeter paper to estimate their body condition using their thorax and abdomen width. The thorax grows only by molting, while the abdomen is more dynamic than the thorax and changes its size during food uptake. As the spiders were fed the same amount during the experiment, the size change due to food uptake is supposed to be similar. The body condition is a better indicator of the nutritional status of a spider than body weight alone because the abdomen is the main food storage and its proportion of the total weight increases with increasing relative width to the thorax width (Anderson, 1974). Furthermore, the body condition controls for the size of spiders, which is important when organisms of different life stages like in our study are compared (Jakob et al., 1996). The program ImageJ version 1.53 k (Rasband, 2018) was used to measure the width. Subsequently, the body condition was calculated as follows (Anderson, 1974):

| Measurement of immune response, dry mass, and PUFA profiles
In the beginning, during and at the end of the experiment, spiders were taken randomly out of the experiment to analyze the immune response, dry mass, and PUFA profiles of spiders. We planned to analyze 10 spiders per treatment and time point, which was hampered by a higher than expected mortality. Nevertheless, at least three spiders were taken per treatment and time point (for exact numbers see Table S2). Additionally, adult chironomid and basic food source samples (Table S2)  of PUFA profiles along the food chain. We sampled chironomids approximately 1 week before the spiders to allow spiders to digest and assimilate PUFA from the consumed chironomids.
To estimate the immune response of the spiders, their ventral abdomen was punctuated with a sterile needle (gauge = 0.45 mm) and subsequently, a nylon monofilament (length ≈ 2 mm, diameter ≈ 0.2 mm) was inserted (Fritz et al., 2017;Rantala et al., 2002;Siva-Jothy & Thompson, 2002). In spiders, the monofilaments cause the same encapsulation reaction as parasites (Ratcliffe & Rowley, 1979). By quantifying the hemocytes adhering to the monofilament, the level of encapsulation can be determined.
24 h after insertion, the spiders were euthanized in liquid nitrogen. The monofilament was recovered from the thawed spiders and stored in ethanol (70%). The spiders were stored at -80°C until further processing. Each monofilament was photographed under a binocular with millimeter paper in three random orientations. In the pictures, the encapsulated area (A encapsulated ) and the area of the nonencapsulated monofilaments (A not encapsulated ) were measured using ImageJ (Rasband, 2018). The ratio of these two areas was reported as proportional encapsulation area (R e/ne ) (Fritz et al., 2017): 18:1n-7 FAME, ALA FAME, Sigma-Aldrich) were used to identify and quantify the FAMEs. The identification was made with OpenChrom version 1.4× (Wenig & Odermatt, 2010). Quantification was conducted in R version 4.2.0 (R Core Team, 2022). Details on the analytical procedure are given in the Appendix S1 under "Analytical procedure PUFA" and on the quantification in the Appendix S1 under "Calculation PUFA content."

| Data analysis
The spiders used at the beginning of the experiment for determining PUFA profiles, dry weight, and immune response were as-  (Benjamini & Hochberg, 1995).
Whenever ANOSIM resulted in significant differences among treatments, similarity percentage (SIMPER) analyses (R-package vegan version 2.5-7, Oksanen et al. (2020)) were performed to identify PUFA contributing to the differences among treatments.
Fresh weight and body condition were analyzed with linear mixed models (LMM) including the explanatory variables time point and treatment as well as their interaction as fixed effects and replicate as random factor because the spider of a replicate was measured repeatedly. The LMM were fitted with the R-package glm-mTMB version 1.1.3 (Brooks et al., 2017). Linear models (LM) with time point, treatment, and their interaction as explanatory variables were used to analyze the dry weight and immune response, with the mean area of the encapsulation and proportional encapsulation area as response variables for the latter. No random factor was required for this analysis because spiders were measured only once for these responses. The effects of treatment, time point, and their interaction on the responses were tested by type II analysis of variance (ANOVA) using χ 2 -test for the LMM and F-tests for the LM. We removed the interaction term from the model when it was not statistically significant. All analyses were made with R version 4.2.0 (R Core

| Contribution of individual fatty acids to treatment differences
SIMPER analysis identified ALA, LIN, EPA, DHA, ɣ-linolenic acid (GLA, 18:3n-6), and linolelaidic acid (LLA, 18:2n-6 t) as main contributors to the differences between treatments (SIMPER, Table 2, Figure 2). In both experiments, ALA and GLA contributed in most cases to the differences between treatments, while LIN, EPA, DHA and LLA explained the differences in only a few cases. In the first experiment, the entire algae treatment (algae, chironomids, and spiders) tended to contain higher levels of ALA than the other treatments (SIMPER,  Figure S3). For spiders between 39% and 43% of the differences in the PUFA profiles were explained by ALA, for chironomids between 49% and 50%, and for the basic food sources between 29% and 38%. The oatmeal treatment differed markedly from the algae and fish food treatments by the GLA content, except once the oatmeal treatments tended to contain more GLA than the other treatments (SIMPER, Table 2). In general, GLA explained between 27% and 45% of the differences in the PUFA profiles between the oatmeal and the other treatments. In the second experiment, the ALA content tended to be higher in chironomids of the leaves treatment than in the chironomids of the fish food and oatmeal treatment.
Furthermore, leaves also tended to contain more ALA then fish food TA B L E 1 Results of the analysis of similarities (ANOSIM) for the polyunsaturated fatty acids (PUFA) profiles.

Experiment
Compared groups R p-Value and oatmeal (SIMPER, Table 2, Figure S4). Overall, ALA explained between 23 and 49% of the differences between the treatments.
The GLA content of the chironomids and base food sources differed markedly between the oatmeal and leaves as well as fish food treatments and explained between 31% and 49% of the differences in the PUFA profiles (SIMPER, Table 2).  (Table 1 and Figure 2).

| Response of fresh and dry weight, growth rate, body condition, and immune response
We aimed to identify a potential influence of dietary PUFA on the physiology and immune response of spiders. We found an effect of treatment only on fresh weight and body condition in the first experiment. The interaction between treatment and time point was significant (LMM, Table 3, Figure 3), whereas during the second experiment, only time point affected the fresh weight of spiders significantly (χ 2 = 12.44, p = .006).
No significant effects of treatment on the immune response and dry weight in the first and second experiment were found.

TA B L E 2 (Continued)
reduced significantly with time in the first experiment (Figure 4), but not in the second experiment (Table 4). Conversely, dry weight was similar across treatment and time in the first experiment, but was reduced significantly with the time (LM: F = 5.71, p = .006) in the second experiment (Table 4).

| Change in PUFA profiles along the food chain
We aimed to identify the influence of dietary PUFA on PUFA profiles along a tritrophic food chain with different basic food sources.
As expected, we found differences in PUFA profiles between treatments at all trophic levels of the food chain, except for the spiders in the second experiment. Temperature in the second experiment was higher than in the first experiment ( Figure S1) and may explain the absence of the treatment effect. Temperature is known to reduce the PUFA content of aquatic organisms as a response to decrease the fluidity of their cell membranes (Arts & Kohler, 2009;Fuschino et al., 2011).
Furthermore, spiders showed high mortality across the experiments, which suggests that the spiders including those surviving were stressed. The stress may have been caused by differences such as the absence of a forested stream in the microcosms compared with their natural habitats resulting in less humidity and shading as well as higher temperatures. Stress requires to invest more energy for maintenance (Calow & Forbes, 1998;Sokolova et al., 2012), thereby reducing the energy available for energetically costly biosynthesis of PUFA Twining, Bernhardt, et al., 2021;Twining, Parmar, et al., 2021) and other physiological processes such as growth (Calow & Forbes, 1998;Sokolova et al., 2012).
In the first experiment, the PUFA profiles of spiders were more similar to the basic food sources than to the chironomids (Figure 2).
Other factors than dietary PUFA uptake can influence the PUFA profiles of spiders. One example is GLA, which was not found in chironomids fed with algae, but in spiders in the algae-based treatment ( Figure S3). The spiders may have stored GLA in their tissue or synthesized it from its precursor LIN (Horrobin, 1992). The latter has not been shown in spiders, but a recent study suggests that spiders are capable of synthesizing EPA from dietary C18 PUFA precursors

| Contribution of individual fatty acids to treatment differences
The PUFA ALA and GLA and the PUFA LIN, EPA, DHA, and LLA were major and minor contributors to the differences between treatments, respectively. In contrast, to another study, which found no GLA in oats (Goedkoop et al., 2007), we found on trend higher GLA levels in oatmeal than in fish food, algae, and leaves. Additionally, the chironomids of the oatmeal-based treatment tended also to contain higher GLA levels then the chironomids of the fish food-, algae-, and leave-based treatment. Therefore, GLA levels in chironomids probably reflect the GLA levels of their diet, which is in line with Strandberg et al. (2020). In the first experiment, spiders of the Note: Degrees of freedom (df), χ 2 and p-value of the ANOVA for fresh weight, growth rate, and body condition of the spiders. Statistical significance is indicated in bold. When the interaction of treatment and time point was not significant, the interaction was not included in the final model.

TA B L E 3
Effects of the explanatory variables treatment and time point as well as their interaction on the fresh weight and body condition of spiders tested with type II analysis of variance (ANOVA) with χ 2 -test for the linear mixed models (LMM).
oatmeal-based treatment also exhibited higher GLA levels than the algae-and fish food-based treatments. This suggests that the spiders GLA levels reflect their diet, though biosynthesis may also play a role as discussed above for the algae-based treatment.
The algae and conditioned leaves had higher levels of ALA than the basic food sources fish food and oatmeal. This is in line with other studies that found ALA in higher amounts in algae than in fish food and oatmeal (Strandberg et al., 2020;Torres-Ruiz et al., 2010). Furthermore, chironomids that consumed algae and Similarly, spiders in the algae-based food treatment displayed higher ALA levels than spiders of the oatmeal-and fish food-based treatments, but the leave-based food treatment did not show different ALA levels. Therefore, nondietary factors may affect the ALA levels in spiders. One factor can be the synthesis of EPA from ALA in spiders . Another factor can be temperature, which was higher during the second experiment with the leaves treatment ( Figure S1). Temperature is known to affect PUFA profiles of invertebrates, as mentioned above. Specifically, ALA levels were shown to decrease with increasing temperature in Daphnia magna Zeis et al., 2019) and terrestrial vertebrates (Hagve et al., 1998;Lund et al., 1999), whereas studies on terrestrial invertebrates, including spiders, are lacking. Additionally, higher temperatures increase the metabolic rate and in turn energy demand of organisms (Brown et al., 2004). As ALA is an energy source for organisms (Tocher, 2003), the spiders may have used ALA to meet their energy demand, thereby decreasing ALA levels.
Anthropogenic stressors such increasing temperatures can have direct effects on the PUFA profiles of organisms, for example, temperature was shown to reduce long-chain PUFA in algae (Hixson & Arts, 2016) and to cause indirect effects by altering species assemblages (Hixson et al., 2015;Martin-Creuzburg et al., 2017) and in turn the availability of PUFA for consumers. Given the wide occurrence of multiple anthropogenic stressors, a realistic assessment for most environmental conditions requires studies that consider the effects of anthropogenic stressors on PUFA transfer and organisms (Kowarik et al., 2022). This is also important in light of vertebrates with a high conservation status, such as birds and bats, potentially relying on longchain PUFA in spiders. These aspects need also to be taken into account to estimate PUFA transfer from aquatic to terrestrial ecosystems.

| Change in PUFA profiles over time
As expected, we found temporal changes in PUFA profiles of the spiders during the experiments. In both experiments, the spiders showed a high variability in initial PUFA profiles, for example in the second experiment two distinct groups were present (Figure 2). This variability may be due to different food sources consumed by the spiders in the field prior to collection. The initial PUFA profiles of the  (Hixson et al., 2015;Martin-Creuzburg et al., 2017;Moyo et al., 2017). Furthermore, spiders are theoretically capable of synthesizing EPA , which may not occur when the spiders retained EPA from their diet prior to collection (Galloway & Budge, 2020). Hence, a sufficiently long experimental duration is required to minimize the effect of the initial PUFA profiles and to detect treatment effects. We addressed this issue by analyzing the PUFA profiles at different timepoints, but the absence of treatment effects on the PUFA profiles of spiders in the second experiment may be explained by an insufficiently long experimental duration owing to the higher mortality.

| Response of spiders fresh and dry weight, growth rate, and body condition to the basic food treatments
We aimed to identify a potential influence of dietary PUFA on the physiology of spiders. We found a treatment effect only in the first experiment, where the interaction of treatment and time point was significant for fresh weight and body condition of spiders, while no effect on dry weight was found. The significant interaction shows that the effect of the treatment on body condition and fresh weight depends on the time point. The PUFA profiles of spiders differed also significantly between treatments. This is in line with Mayntz and Toft (2001), who showed that PUFA enhanced fresh weight of spiders.
By contrast, in the second experiment, fresh weight and dry weight differed between time points but not between treatments, while body condition was similar across treatments and time points. Furthermore, the PUFA profiles of the spiders were similar across treatments in the second experiment. The different response of spiders across the experiments may be explained by the study durations and conditions such as temperature. The first experiment lasted for approximately 6 weeks, whereas the second microcosm experiment was terminated after 3 weeks. A longer study duration may have resulted in detectable treatment effects also in the second experiment. Furthermore, the temperature during the second experiment was higher (Figure S1), and it was shown that with increasing temperature, the requirements of organisms for PUFA can be reduced (Masclaux et al., 2009)

| Immune response of spiders to the basic food treatments
Contrary to our expectation, the immune response of spiders was only affected significantly by time point and not by treatment. This is in contrast to a field study, in which the long-chain PUFA EPA and DHA were linked to enhanced immune responses of spiders (Fritz et al., 2017). In our study, food sources of spiders contained only low levels of these long-chain PUFA, which may explain the absence of a treatment effect. Nonetheless, the immune response is affected by many factors not considered in our experiments, which may explain our finding. For instance, the immune response can decrease with reduced food intake (Siva-Jothy & Thompson, 2002) and changes in the dietary composition (Srygley et al., 2009). In our study, spiders received only one food source. In real-world ecosystems, spiders consume and benefit from multiple prey types (Nyffeler, 1999;Uetz et al., 1992). That is because a balanced nutrient composition of prey is more important for the performance, for example, survival of spiders, than single nutrients (Mayntz & Toft, 2001). Additionally, depending on their hunting strategy, spiders are capable to obtain an optimal nutrient composition through adjusting foraging strategies. For example, mobile spiders such as ground hunters are capable to choose prey actively (Mayntz et al., 2005). Future studies that also consider other factors such as nutrient availability and anthropogenic stressors can help to estimate the importance of PUFA for immune response in relation to other factors.

| CON CLUS IONS
PUFA can affect the weight and body condition of spiders, where this depends on the environmental context. This context includes, among others, diverse food sources, that is, several prey types, food chains with interactions between taxa, long duration for PUFA assimilation, a range of environmental factors (e.g., temperature), and anthropogenic stressors.
Furthermore, the PUFA profiles can differ across trophic levels for multiple food sources. Aquatic and semi-aquatic food sources may result in more distinct PUFA profiles of chironomids and spiders than terrestrial food sources. ALA and GLA are among the major contributors to these differences in PUFA between food sources.
However, environmental factors such as temperature also influence PUFA profiles. Future studies under more realistic conditions are needed to improve our understanding of the effect of PUFA in ecosystems and to evaluate the transferability of our results.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

O PE N R E S E A RCH BA D G E S
This article has earned an Open Data badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://doi.org/10.5281/ zenodo.7692685.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available Sexual advertisement and immune function in an arachnid